Patients treated with autologous CLL cells transduced with an adenovirus encoding murine CD154 (Ad- CD154) experienced acute declines in leukemia cell counts and lymph node size and long-term desirable effects, including increased numbers of leukemia-reactive T cells, generation of anti-CLL antibodies, and apparent slowing or halting of disease progression. Treated patients had changes in bystander nontransfected CLL cells, reflecting activation by CD154 in vivo. We found that CD154-activation induced a programmed series of events in CLL cells, which initially were transiently protected and then sensitized to apoptosis induced by ligation of induced extrinsic death receptors (e.g. CD95 and DR5), cytotoxic drugs, or novel agents that inhibit the inhibitor of apoptosis (IAP) proteins (see project 2). Sensitivity to these agents appears associated with the capacity of CD154-activation to induce expression of the pro-apoptotic molecule Bid through a p53-independent, c-Abl-dependent mechanism, which apparently involves the alpha isoform of p73. This raises the exciting prospect that Ad-CD154 gene therapy may circumvent the dependency on p53 of anti-leukemia drugs, while inducing anti-leukemia cellular and antibody immune responses. The latter already have allowed us to identify a novel CLL-associated antigen, Ror1, which potentially could be used in assays to monitor the activity of Ad-CD154 gene therapy or in vaccines for immune therapy of this disease. For future clinical studies, we developed a "humanized" CD154 (designated ISF35) that can be stably expressed at high-levels on the CLL plasma membrane. This project has the following specific aims: (1) Continue ongoing analyses of the mechanism(s) responsible for the acute fall in leukemia-cell counts observed in patients treated with autologous Ad-CD154-transduced, or ISF35-transduced, CLL cells;(2) Examine cellular immune responses against a newly-identified CLL-associated antigen, Ror1;(3) Evaluate the specificity and biologic activity of anti-leukemia antibodies induced by Ad-CD154 gene therapy, with special emphasis on anti-Rorl autoantibodies. (4) Develop the E mu-TCL1 mouse model generated in Project 1 to evaluate parameters intended to maximize the activity of Ad-CD154 gene therapy, either alone or in combination with anti-leukemia drugs or immune-based treatment strategies.